PROJECT SUMMARY At least 300,000 people are living with thyroid cancer in the United States, and more than 1,500 of them die each year from this disease. Oncogenic alterations in BRAF, RET/PTC, RAS, and PIK3CA are common in many thyroid cancers, and targeted therapies against these pathways are being tested. However, initial trials targeting this pathway have not been as impressive as expected at this stage. Thus, it is critical to further elucidate the molecular mechanisms of thyroid cancer progression and metastasis and to identify novel drug targets. Focal Adhesion Kinase (FAK) is overexpressed and activated in numerous tumor types and has emerged as a promising therapeutic target, especially in relation to metastasis. FAK is a multifunctional kinase whose activity is dependent on Src kinase and upon full activation the FAK-Src signaling complex regulates multiple cellular responses including proliferation, invasion, and metastasis. We recently discovered that FAK is overexpressed and activated in thyroid cancer clinical samples as well as cell lines derived from advanced thyroid cancer patients. We further demonstrated that the growth and invasion of thyroid cancer cells expressing high levels of phosphorylated FAK are susceptible to treatment with the Src inhibitor, saracatinib, which was used to inhibit FAK function. Notably, levels of activated Src did not correlate with Src inhibitor sensitivity, suggesting that FAK is the key mediator of oncogenic signaling in this pathway. Recently, agents targeting FAK have entered clinical trials, providing the first opportunity to directly target the kinase activity of FAK. The specific role of FAK in thyroid cancer growth, invasion, and metastasis has not been tested. FAK functions as a signaling kinase and a scaffolding protein, complicating its role as a clinical target. Our preliminary data using pharmacologic and genetic approaches to block the kinase and/or scaffolding functions of FAK, suggest that these dual functions may regulate different cellular processes in thyroid cancer. The goals of this proposal are to understand the regulation and function of FAK as a central mediator of these pro- tumorigenic processes. In Aims 1 and 2, we will use genetic shRNA FAK knockdown in parallel with pharmacologic approaches with recently developed FAK inhibitors to define the specific role of the kinase and scaffolding functions of FAK in the growth, invasion, and survival of thyroid cancer cells in vitro (Aim 1); as well as in vivo using a novel orthotopic thyroid cancer model and an experimental metastasis model, which we developed (Aim 2). In Aim 3, we will validate the clinical relevance of FAK signaling using a comprehensive thyroid tumor microarray. Successful completion of these aims will determine whether inhibition of FAK represents a rational and more specific therapy for patients with advanced thyroid cancer and the prevention of metastases as well as other poorly differentiated cancers with oncogenic FAK signaling.